The efficiency of MSC‐based targeted AIE nanoparticles for gastric cancer diagnosis and treatment: An experimental study

Abstract Mesenchymal stem cells (MSCs), due to their tumor tropism, are strongly recruited by various solid tumors and mobilized by inflammatory signals in the tumor microenvironment. However, effective cellular uptake is critical for MSC‐based drug delivery. In this study, we synthesized a spherical copolymer, polyethylenimine–poly(ε‐caprolactone), with aggregation‐induced emission (AIE) material and the anticancer drug, paclitaxel, coloaded onto its inner core. This was followed by the addition of a transactivator of transcription (TAT) peptide, a type of cell‐penetrating peptide, to modify the nanoparticles (NPs). Finally, the MSCs were employed to carry the TAT‐modified AIE‐NPs drug to the tumor sites and assist in simultaneous cancer diagnosis and targeted tumor therapy. In vitro, the TAT‐modified AIE‐NPs showed good biocompatibility, targeting, and stability in an aqueous solution besides high drug‐loading and encapsulation efficiency. In vitro, the AIE‐NPs exhibited a controllable release under a mildly acidic environment. The in vivo and in vitro studies showed high antitumor efficacy and low cytotoxicity of the AIE‐NP drug, whereas biodistribution confirmed the tumor tropism of MSCs. To summarize, the MSC‐based AIE‐NP drugs loaded with TAT possessed good biocompatibility and high antitumor efficacy via the enhanced NP‐drug uptake. In addition, the tumor tropism of MSCs provided selective drug uptake by the tumor cells and thus reduced the systemic side effects.

cells by prolonging circulation time and increasing drug retention in tumor tissues, attributed to the impacts of enhanced permeability and retention. 4 By modifying the surface of nanoparticles (NPs), nanocarriers can precisely deliver drugs to the cancer cells, release them in a controlled manner, and decrease off-target toxicity.
Liposome-encapsulated doxorubicin (Doxil) and PEG-L-asparaginase (Oncospar) are the two successful representatives used in clinics. 5,6 Many strategies such as stimuli-responsive nanocarriers have been used to improve delivery efficiency 7,8 ; however, the rapid recognition and clearance of nanocarriers from the bloodstream by the reticuloendothelial system (RES) limit their usefulness as drug carriers.
Previous research showed that mesenchymal stem cells (MSCs) are strongly recruited by various tumors and mobilized by inflammatory signals from the tumor microenvironment. [9][10][11] The natural tumor tropism of MSCs and their ability to inoculate into cancer sites, along with their low immunogenicity, stability, and expandability, make them the ideal drug carrier for the delivery and improved bioavailability of anticancer agents. 12,13 The MSC-based vehicles with drug-loaded NPs provide an effective alternative to overcome the biodistribution limitations of NPs; the ability of MSCs to migrate within the tumor tissue protected the NPs from clearance by the body's immune system and enabled their entry into the tumor core. 8 Studies have demonstrated that MSCs loaded with chemotherapeutic NPs homed to tumor sites and achieved cellular drug storage, which release the drug in a controlled manner in prostate, lung, and glioma animal models. 14,15 The imaging technique helps in the early diagnosis of cancers and tracking of real-time drug release. Fluorescence imaging has been used widely in the biomedical field due to its rapid signal acquisition and high detection sensitivity 16 ; however, the imaging performance of traditional fluorescent dyes incorporated in NPs at high loadings is limited by the aggregation-caused quenching (ACQ) effect. 17 The aggregation-induced emission (AIE) phenomenon discovered by Tang et al. addressed the ACQ drawbacks. 18 The AIE-active materials have been used in bioimaging and drug delivery because they are nonluminescent in dilute solutions and become highly emissive when aggregated. 19,20 Tetraphenylethylene (TPE), an AIE fluorogen, S C H E M E 1 Schematic illustration of the MSC-based NPs drug platform for targeted cancer therapy. The TAT peptide facilitates MSC and tumor cell uptake of the NP drugs. After the intravenous injection, the inherent tumor tropism of MSCs can target the tumor tissue and release the therapeutic cargo through exocytosis. Subsequently, the NP drug can be internalized by the tumor cells and released under the acidic environment of endo/lysosomes, inducing efficient apoptosis of the tumor cells. MSC, mesenchymal stem cells; NP, nanoparticle; TAT, transactivator of transcription possesses high biocompatibility and has been used extensively in bioimaging and drug delivery. 21 This study used a novel MSC-based platform with TPE loaded onto the copolymer polyethylenimine-poly (ε-caprolactone) (PCL-PEG), packeted with paclitaxel (PTX), and utilizing a cell-penetrating peptide to modify the drug-loaded NPs to enhance its cellular uptake (Scheme 1). We analyzed the physical and chemical characteristics of the drug-loaded AIE-NPs, biosafety of the synthesized NPs copolymers, and evaluated the uptake efficacy and

| Synthesis of PCL-PEG
PCL-PEG was synthesized by ring-opening polymerization as described previously with slight modifications. 22 First, the initiator benzyl alcohol was exploited for the synthesis of monohydroxyterminated poly(ε-caprolactone) (PCL-OH), with SnOct (0.1% moles of ε-caprolactone) as a catalyst. Then, 4-nitrophenyl chloroformate (NPC) (1.2 g, 3 mmol) and pyridine (0.4 ml, 5 mmol) were added to PCL-OH (1.2 g, 0.6 mmol), dissolved in dichloromethane, and the reaction was carried out under the nitrogen atmosphere at room temperature for 24 h. Subsequently, we purified PCL-NPC via precipitation in cold ether and dried it under vacuum. Afterward, THF solution (2 ml) containing PCL-NPC (0.2 g, 0.1 mmol) and DMSO solution (5 mL) containing PEG-5 k (1 g, 0.5 mmol) were mixed and agitated for 24 h at room temperature. Finally, ether was added into the above reaction solution to extract PCL-PEG and vacuum dried.

| Synthesis of PCL-PEG-TAT
The TAT peptide stock solution was prepared with 12.28 mg of TAT dissolved in 200 μl of DMSO, followed by the addition of EDC and NHS to activate the TAT peptide for 4 h. Later, THF (10 ml) containing PCL-PEG-NH 2 (20 mg) was dropped into the activated TAT peptide solution and maintained reaction for another 24 h. The PCL-PEG-TAT copolymer conjugates were then obtained by precipitating the mixture with cold ether. The solid product was washed three times and preserved at 4 C for subsequent use.

| Preparation of PCL-PEG-TAT/PTX/TPE
To prepare the PCL-PEG-TAT/PTX/TPE nanomicelles, the thin-film hydration method was utilized. 23 A small amount of PCL-PEG-TAT (30 mg), PTX (10 mg), and TPE (10 mg) were dissolved in dichloromethane (20 ml), and the mixture was placed in a rotary evaporator to obtain a dry film at 45 C under vacuum. Water (10 ml) was input into the dry film while the solution was vortexed and sonicated for 30 min. The obtained micelle was filtered through a millipore filter with a pore size of 0.8 μm to remove the unencapsulated PTX and TPE. The PTX-loaded PCL-PEG-TAT/TPE micelles were conserved at 4 C for further experiments. The PCL-PEG/PTX/TPE was prepared as mentioned in Section 2.2.2, but with PCL-PEG instead of PCL-PEG-TAT.

| Encapsulation efficiency and loading capacity
A small amount (10 mg) of PCL-PEG/TPE/PTX was deliquesced in DMSO and diluted to a final volume of 10 ml. Centrifugation The cytotoxicity of blank PCL-PEG-TAT copolymer was evaluated similarly using the SGC-7901 cells and MSCs with a copolymer concentration gradient from 1 to 320 μg/ml. Cell viability was computed by applying the formula below: Cell viability % ð Þ¼ OD of the experimental group À OD of the blank group ð Þ = OD of the negative control group À OD of the blank group ð Þ Â 100%:

| Cell endocytosis study
The efficacy of TAT in mediating cellular uptake of PCL-PEG/TPE was investigated. The MSCs with a density of 5 Â 10 4 cells per well were seeded in a 24-well plate and incubated overnight to allow attachment at 37 C under 5% CO 2 . Afterward, the cells were exposed to a

| Intracellular distribution
We further studied the intracellular distribution of PCL-PEG-TAT/ TPE. The MSCs were exposed to 20 μg/ml PCL-PEG-TAT/TPE for 0.5, 2, 4, and 6 h. Then, MSCs were washed with PBS (2 ml) and stained with Lyso-Tracker Green (75 nM) for 1 h. The cells were then fixed with 4% paraformaldehyde for 30 min, followed by a PBS wash.
A confocal laser scanning microscopy (CLSM) (LSM880; Zeiss) was used for observing the intracellular distribution of PCL-PEG-TAT/TPE.

| Intracellular drug retention in MSCs
The

| In vivo biocompatibility evaluation
The toxicity of different formulations was also investigated in vivo. After 21 days of treatment, blood samples of each group were collected through retro-orbital plexus into centrifuge tubes and rested at room temperature for 2 h. Afterward, centrifugation was used to separate the serum from the cells (3000 rpm, for 5 min). The upper serum was kept at À80 C for subsequent evaluation of biochemical parameters, including enzyme activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and values of albumin (ALB), creatinine (CREA), uric acid (UA), urea (UREA), and glucose (GLU). Euthanasia was used for the mice, and their vital organs, including the heart, liver, spleen, lungs, and kidneys, were dissected for tissue analysis by the H&E staining.

| Statistical analysis
Results are represented as mean ± SD. For each group, at least three replicates were set, and two independent experiments were F I G U R E 1 (a) 1  The smaller size observed from the TEM photographs was presumably due to dehydration of the NP copolymers. The morphology, size, and Pdl indicate that the PTX-loaded copolymers had good aqueous dispersibility, stability, and uniformity. The mean diameter of such NPs was less than 200 nm, making them easier to be engulfed by MSCs. 13.64% and 63.82%, respectively. The internal hydrophobic structure of PCL-PEG contributes enough space to load PTX, which helps achieve high drug-loading efficacy. Figure 2c shows the release curve of free PTX and the cumulative release curve of PTX from PCL-PEG/ TPE/PTX in PBS (pH 5.7). The free PTX was released at a zero-order release phase, whereas the PTX from NP complex was released at a relatively fast rate for the first 20 h; a cumulative release of 41.5%.

| AIE behavior
Then, the release rate gradually slowed down, and the cumulative release at 98 h was 60.49%. The initial rapid release of PTX from the NPs at pH 5.7 was partially due to the rapid degradation of the PCL-PEG copolymers under acidic conditions and partially attributed to the carboxylic groups' protonation in the PCL-PEG copolymers, which weakened the electrostatic interactions between the cationic PTX and the anionic polymer carrier. 32,33 Intensely hydrophobic interaction between PTX and PCL led to the slow-release phase. 34 The rapid release under a lower pH condition favors the release of PTX from NPs in the acidic environment of the endo/lysosomes in the tumor cells, thus exerting a therapeutic function.

| In vitro cell cytotoxicity analysis
The SGC-7901 and MSCs viability after incubation with various condensations of PCL-PEG-TAT were gauged by the CCK-8 assay

| Drug retention in MSCs
The drug retention of NPs in cells mainly depends on the efflux of cells and the dilution effects of cell division. We hypothesize   The classical anticancer agent, PTX, promotes microtubule assembly, inhibits cell proliferation and apoptosis induction. 36 In this study, Annexin V-PE/7-AAD double staining was adopted to evaluate the apoptosis-inducing capability of different agents on SGC-7901 cells. PBS was used as the control. The flow cytometry scatter plot shows the apoptosis rate of SGC-7901 cells to be 32% when treated with PCL-PEG/TPE/PTX. The rate increased slightly to 38% when MSCs were utilized as an NP drug carrier, and the rate was further elevated to 47% when TAT was introduced due to its enhanced drug intake by the SGC-7901 cells (Figure 5b with previous studies, 37 we also observed that the drug-loaded NPsprimed MSCs remained in the lungs due to mechanical entrapment within the lung capillary beds; however, the rapid entrapment dis-

CONFLICT OF INTERESTS
The authors declare no conflict of interests.

DATA AVAILABILITY STATEMENT
Data openly available in a public repository that issues datasets with DOIs.